Recombinant lactoferrins, methods of production from plants and uses thereof

ABSTRACT

The invention concerns the use of a recombinant nucleotide sequence containing a cDNA coding for a lactoferrin, in particular human lactoferrin, or the derived proteins, and elements enabling a plant cell or produce lactoferrin or the derived proteins, coded by said cDNA, in particular a transcription promoter and terminator identified by the plant cell transcription machinery, to transform plant cells in order to obtain, from these cells, or plants obtained therefrom, lactoferrin or derived proteins.

[0001] The present invention relates to recombinant lactoferrins (rLf),their production from plants, and uses thereof.

[0002] The use of certain proteins found in mammals has been questioneddue to the possibility of contamination by non conventional infectiousagents, particularly of the prion type. The impact on marketing andregulations is great. The development of proteins free of all animalcontamination is, therefore, a new possibility but one which faces newdifficulties depending on the protein and the plant matter chosen.

[0003] Prior art includes the publication by Mitra et al. (1994), whichdescribes the transformation of tobacco cells with the DNA sequencewhich codes for human lactoferrin. However, this publication is limitedto plant cells, and does not allow for the production of regeneratedplants from these cells. Furthermore, the protein expressed is notpurified, and seems, in any case, imperfect as only a part (48 kDa) isdetected, and not the entire protein.

[0004] In the prior art, application WO 9637094 is also known; thisdescribes the production of plants which are resistant to viruses, bymeans of transforming them with a gene which codes for lactoferrin.However, in this document as well, the protein is not purified and theonly indication of its presence is a Weston blot test.

[0005] As a result of the complexity of lactoferrin and thecharacteristics of the plant matter used, the extraction andpurification procedures also represent major obstacles in the productionof lactoferrin from plants. The difficulties posed by these twoprocedures can represent a new set of problems for each protein whichone wishes to produce from a plant.

[0006] Lactoferrin is a glycoprotein of the transferrin family.Following its discovery in human milk, lactoferrin was shown to bepresent in many other species such as cows, goats, pigs, mice and guineapigs, but a highly variable concentrations. In human milk, theconcentration of lactoferrin is in the range of 1 to 2 g/l; theconcentration is particularly high in colostrum and diminishes over thecourse of lactation. In milk, lactoferrin is present primarily in theapo form, that is to say, unsaturated in iron. Lactoferrin has also beenfound in many other secretions, such as the saliva, bile, pancreaticfluid, and secretions of the small intestine. It is found in most mucus,such as bronchial, vaginal, nasal and intestinal secretions.

[0007] Lactoferrin is also present in polymorphonuclear neutrophilicleucocytes, where it is localized in the secondary granules of cellsthat do not contain myeloperoxidase. Leucocytic lactoferrin issynthesized during granulopoiesis of the promyelocyte stage of thenmetamyelocyte stage. When the neutrophils degranulate, lactoferrin isreleased into the plasma at a relatively low concentration (0.4 to 2mg/l) as compared with the level of transferrin found in the blood (2 to3 g/l).

[0008] The peptide sequence for human lactoferrin (hLf) was determinedin 1984 by Metz-Boutigue et al. This sequence of 692 amino acids wasconfirmed by way of cloning of the cDNA for lactoferrin of the humanmammary gland (Powell and Ogden, 1990, Rey et al., 1990). Lactoferrinand serotransferrin have very similar primary structures and spatialconfigurations. Their polypeptide chains are formed of two lobes (Nterminal lobe and C terminal lobe) joined by a small alpha helixpeptide. Sequence similarities between the N and C terminal halves ofhuman lactoferrin reach 37%. Trypsic hydrolysis of human lactoferrinallowed Legrand et al. (1984) to produce the 30 kDa N trypsic (N-t)fragment (residues of amino acids 4 to 283), and the 50 kDa C trypsic(C-t) fragment (residues of amino acids 284 to 692). At equimolarproportions, these fragments can reunite to form a non-covalent N-t/C-tcomplex which has electrophoretic and spectroscopic characteristicssimilar to those of human lactoferrin (Legrand et al, 1986).

[0009] Lactoferrin can reversibly bind two ferric ions which results ina salmon-pink coloration, the maximum absorption of which is centered at465 nm. The binding of each ferric ion requires the same of a carbonateion. At a pH of 6.4, the association constant of the complex [Fe3+]2-Lfis in the range of 10²⁴ M⁻¹, which decreases with, pH. X-ray diffractionstudy of human lactoferrin at 3.2 Å to 2.8 Å and at 2.2 Å show that eachferrous ion is coordinated with 2 tyrosine residues, a histidine, anaspartic acid and a carbonate ion. These amino acids are the same inboth lobes. Both lactoferrin iron binding sites have a strong affinityfor this metal, but they release it at different pH levels. The N-t lobereleases its iron at pH 5.8 (acid labile lobe), while the C-t lobe (acidstable lobe) releases its iron at pH 4. Other ions may bind to theprotein, in particular gallium. Researchers interested in the use of a⁶⁷Ga-Lf complex as a tracer in cancer diagnostics have shown thatfollowing injection with ⁶⁷Ga, the complex is preferentially found inthe mammary tissues, in physiological and pathological secretions and inBurkitt and Hodgkin lymphomas.

[0010] In terms of glycosylation, lactoferrin isolated from human milkhas three glycosylation sites; Asn¹³⁸, ASN⁴⁷⁹ AND Asn⁶²⁴, the firstlocated on the N-t lobe and the other two on the C-t lobe. Glycosylationoccurs preferentially at two sites (Asn¹³⁸ and ASN⁴⁷⁹) in 85% ofmolecules, while glycosylation of one site (Asn⁴⁷⁹) and of the threesites simultaneously happens in 5% and 9% of cases, respectively.Lactoferrin glycans are of the mono or disialylated and fucosylatedN-acetyllactosamine type (Spik et al, 1982). The fucose residues are α(1,6) branched on the N-acetylglucosamine 5′ of the attachment point, orare α (1,3) branched on the N-acetylglucosamine 5′ of the antenna.Leucocytic lactoferrin differs from that above by the total absence offucose.

[0011] While serotransferrin is unquestionably the principal transporterof iron in all the cells of the organism, the roles played bylactoferrin appear to be essentially linked to defense of the organismand inflammatory mechanisms, working either directly on pathogenicmicro-organisms, or indirectly on the effector immune cells.

[0012] Lactoferrin is an anti-microbial agent which works by way ofseveral mechanisms. The first of these is the bacteriostatic effect oflactoferrin by means of iron deprivation (Spik et al., 1978). By takingup iron from its surroundings, lactoferrin inhibits bacteria division,as iron is an indispensable element in the biosynthesis of DNA.Furthermore, a more complex mechanism which causes antibodies to act hasbeen shown. The bacteriostatic activity of lactoferrin in creases in thepresence of the specific IgA and IgG of the pathogenic bacterium. At thesame time, lysozyme can associate its lytic activity on the walls ofGram+ bacteria with the action of lactoferrin. Thus, in milk,lactoferrin, lysozyme and antibodies can act synergistically in case ofmicrobe attack.

[0013] The second anti-bacterial effect of lactoferrin is linked to itbactericidal capacity. Lactoferrin appears to bind to the walls ofGram-bacteria, which appears to destabilize them and to provoke therelease of lipopolysaccharides (LPS). Thus it would seem that the wallsbecome more fragile and more susceptible to the effects of hydrophobicantibiotics. These theories have been: confirmed by use of electronmicroscopy which shows the destabilizing effects of lactoferrin onGram-bacteria, including E. coli. A hypothesis has been made to theeffect that the binding of lactoferrin occurs on the A lipid of the LPS,and that this is followed by the extraction of these LPS from theexternal membranes of the bacteria, irreparably damaging them. Thebactericide regions of human lactoferrin (lactoferrin A) and bovinelactoferrin (lactoferrin B) have recently been identified. They are bothfound in an N-terminal lobe loop comprising 18 amino acids. This loop isformed by a disulphur bridge between the residues Cys 30 and 37 forhuman lactoferrin, and 19 and 36 for bovine lactoferrin. In addition,the importance of the loop formed by residues 28-34 of human lactoferrinin its binding to LPS has been demonstrated.

[0014] Thus, due to its bacteriostatic and bactericide activities,lactoferrin present in human milk protects nursing infants frominfantile diarrheas.

[0015] An anti-fungal effect has been established for lactoferrin withrespect to several strains of Candida. Studies have also been carriedout with bovine lactoferrin showing this action both on yeasts and onfilamentous fungi. Also, the effect of bovine lactoferricin appears tobe greater than that of whole bovine apolactoferrin and similar to thatof polymyxin B, an antibiotic of the cationic peptide type, known forits membrane destabilisation properties. It has also been shown thatlactoferrin B interacts directly with the surface of the fungus, therebyintroducing changes to its ultrastructure.

[0016] Recently, an antiviral activity has been demonstrated forlactoferrin by several authors. Certain types of virus penetrate thecells by means of a mechanism which causes absorption of proteoglycansby the membranes of the target cells, followed by binding at a specificreceptor and fusion of the viral membrane with that of the host. As itis highly basic, lactoferrin binds to the heparan sulphates of cells,and is thereby able to inhibit the adsorption of several types of virus.Highly conclusive in vitro studies have been carried out using HIV(human immunodeficiency virus) and HCMV (human cytomegalovirus) with aCI50 in the range of 10 μg/ml. Similar results have been achieved withHSV-1 (herpes simplex virus type 1). This work showed not only blockingof virus receptors (heparan sulphates, proteoglycans, LDL receptor), butalso a possible interaction between lactoferrin and the virus. Theserecently discovered roles represent new possibilities for prevention andtreatment, particularly in terms of immunodeficiency illnesses orrecurrent illnesses, in terms of these viral infections.

[0017] One of the consequences of tissue inflammation is the formationof free radicals and the peroxidation, of lipids. The formation of thesefree radicals results particularly from the phagocytosis mechanisms ofthe micro-organisms. According to the Haber-Weiss reaction, freeradicals are created as the result of ferric iron, which acts as areaction catalyst. It has been shown in vivo that lactoferrin, comingfrom the degranulation of neutrophils, stops the formation ofextra-cellular free radicals by immediately taking up the iron whichwould catalyse the formation of these radicals. As the result of thisaction, lactoferrin prevents tissues from being damaged. By means of asimilar mechanism, lactoferrin inhibits the peroxidation of lipids andthus protects the cellular membranes to which it binds. These studiesalso showed that lactoferrin has anti-oxidising and anti-inflammatoryeffects if the protein is in the form without iron.

[0018] Lactoferrin released by leucocyte granules has been identified asan inhibitor of granulocyte and macrophage colonies by reducing theproduction of GM-CSF (granulocyte and macrophage colony stimulationfactor). The mechanism involved seems complex, as lactoferrin appears toact by inhibiting the release of a monokine, which is itself responsiblefor the release of GM-CSF by the lymphocytes, the fibroblasts and theendothelial cells. This monokine has been identified at IL1.

[0019] Many of the roles played by lactoferrin, such as the suppressionof the production of antibodies, regulation of complement activation andregulation of NK (natural killer) cells activity, suggest mechanismswhich are regulated by cytokines. Studies show that lactoferrin canexert a negative retrocontrol on certain cytokines such as the IL1, theIL2 and the TNFa, so as to prevent the activation of leucocytes ininflammation areas.

[0020] Zimecki et al (1991) showed that immature CD4-CD8-thymocytesincubated in the presence of lactoferrin acquire the marker CD4+characteristic of auxiliary lymphocytes. These authors also point outvarious phenotypic and functional changes in B cells in the presence oflactoferrin (Zimecki et al., 1995). A specific receptor for humanlactoferrin has been defined for activated lymphocytes (Mazurier et al.,1989). In addition, the site of interaction between lactoferrin and itslymphocytic receptor has been described: Legrand et al. (1992) showedthat it was contained in the N terminal region, and more specifically,in the first 50 residues of lactoferrin.

[0021] Lactoferrin appears to regulate the cytotoxic activity of NK(natural killer) cells and LAK cells at very low quantities (0.75μg/ml). Lactoferrin significantly increases the cytotoxic activity of NKcells with respect to tumour cells and cells infected by retroviruses.Lactoferrin also effects, the cytotoxicity of monocytes. The cause ofthis stimulation by lactoferrin of the cytotoxic activity of NK cells,of lymphocytes and also adherent cells may be the result of eitheractivation of the killer cells following the internalisation oflactoferrin, or of a modification of the target cells which then becomemore susceptible to lysis.

[0022] Lactoferrin also seems to play a immunoregulatory role ininflammatory responses (Elass-Rochard et al., 1995; confirmed byElass-Rochard et al., 1998). Demonstration of anti-tumour activity wasalso the object of studies by Salmon et al (1998).

[0023] Lactoferrin has a growth factor effect on various cells inenvironments lacking in foetal veal serum. This activity has beendemonstrated, particularly on the B and T lymphocytic strain withrespect to a macrophage murine strain (P388 DI). Lactoferrin alsostimulates the incorporation of thymidine tritiate in the DNA of ratenterocytic cells.

[0024] The role of iron in growth factor activity is still an object ofcontroversy. According to some authors, lactoferrin works by providingthe iron necessary to cellular proliferation. Others hold that iron isnot involved, and that the mitogen activity is due solely to the proteinitself.

[0025] The hypothesis to the effect that lactoferrin is involved in theintestinal absorption of iron results from the observation that amongstbreast-fed infants, the incidence of iron deficiency is very low. Infact, only such children maintain a major supply of iron up to the ageof 6 months, which implies high bioavailability for iron contained inhuman milk. The percentage of absorption can reach 81% during the firstthree months of life, and diminishes rapidly thereafter. Amongst thevarious components of human milk, lactoferrin is the best candidate forexplanation of both the great bioavailability of iron in milk andregulation of its absorption. Cox et al studied the importance oflactoferrin in the absorption of iron by the human intestine as early as1979. The enterocytic receptor for lactoferrin was first shown to bepresent in rabbits, then in mice. Finally, the human enterocyticreceptor was studied. It was shown that, with HT29 enterocytic cellcultures, the number of lactoferrin receptors doubled in the presence ofiron chelator (Mikogami et al., 1994, 1995). This increase is due to ade novo synthesis of receptors. The expression of the lactoferrinreceptor is, therefore, regulated by a lack of iron, and this lack alsoinduces an increase in the internalisation of the lactoferrin receptorby the enterocytes. Thus, lactoferrin seems to be involved in ironnutrition, in particular, in case of iron deficiencies.

[0026] Not only has the cDNA sequence for human lactoferrin beendetermined, but the cDNA sequence for various animal species has beenestablished. Specifically, sequences have been found for:

[0027] bovine lactoferrin (Pierce et al., 1991)

[0028] porcine lactoferrin (Alexander B. F. et al., 1992)

[0029] murine lactoferrin (Shirsat N. V. et al, Gene, 1992)

[0030] caprine lactoferrin (Le Provost F. et al, 1994).

[0031] Analysis of peptide sequences corresponding to cDNA sequencesindicates major similarities. In particular, the human lactoferrinsequence shows a 69% correspondence with that of cows. As with humanlactoferrin, the nucleotide sequence of bovine lactoferrin cDNA containsa signal peptide composed of 16 amino acids.

[0032] This structural similarity results in functional similarities. Inparticular, as the N-terminal end of both bovine and human lactoferrinhave a great number of basic amino acids, these two lactoferrins arerecognised in a similar way by many constituent acids. It should benoted, for example, that these two proteins have similar interactionswith the lipopolysaccharides of Gram-bacteria and the proteoglycanswhich are found on the surface of many cells. (Elass-Rochard E., et al.,1995).

[0033] These interactions appear to imply that these two lactoferrinsmay play the same role in the inhibition of release of cytokines ofmacrophages activated by lipopolysaccharides, and therefore ininflammatory reaction mechanisms.

[0034] In the same manner, these two lactoferrins show the same bindingcharacteristics for enterocytic cells and, therefore, play the same rolein intestinal iron absorption and various anti-bacterial and anti-viralactivities.

[0035] Thus, the many roles played by lactoferrin, and especially itsantioxidant, anti-microbial and anti-viral activities make this proteinof major importance in terms of treatment, and above all prevention, ofbacterial, fungal and viral infections, and also in terms of theprevention of septic shock which, classically, occurs after surgicaloperations.

[0036] The immunoregulatory and anti-tumour activities of lactoferrincan also be used in the treatment of inflammation and cancer (Denis etal.; Zimecki et al.).

[0037] Lactoferrin can also be used in dermo-pharmaceutical and cosmetictreatments, for example in a cosmetic anti-free-radical treatment, or toprotect hair

DETAILED DESCRIPTION OF THE INVENTION

[0038] The invention concerns:

[0039] the use of a recombinant nucleotide sequence containing both: acDNA coding for a lactoferrin, particularly for mammalian lactoferrin,preferably bovine, porcine, caprine or human lactoferrin, or derivedproteins (derived protein meaning any protein having at least 70%correspondence with the target protein, particularly at least 80%, forexample between 85 and 100% correspondence and/or having a differentglycosylation profile wile maintaining the functional characteristics ofthe reference lactoferrin); and the elements required by a plant cell toproduce the lactoferrin or derived proteins coded by said cDNA,particularly a transcription promoter and terminator recognised by thetranscriptional machinery of plant cells, so as to produce from thesecells, or from plants produced therefrom, lactoferrin or derivedproteins.

[0040] a recombinant nucleotide sequence: characterised in that itcontains both: a sequence coding for lactoferrin, particularly formammalian lactoferrin, preferably bovine, porcine, caprine or, humanlactoferrin, or derived proteins; and the elements required by a plantcell to produce the lactoferrin or derived proteins coded by said cDNA,particularly a transcription promoter and terminator recognised by thetranscriptional machinery of plant cells. Advantageously, the nucleotidesequence contains a sequence coding for a signal peptide responsible forthe secretion of recombinant polypeptides.

[0041] a vector, particularly a plasmid, containing a nucleotidesequence according to the invention inserted, as necessary, at a sitewhich is not essential for its replication.

[0042] a cellular host, particularly any bacteria such as Agrobacteriumtumefaciens transformed by a vector according to the invention,

[0043] a method of producing lactoferrin, particularly humanlactoferrin, or derived proteins, characterised in that it comprises:

[0044] transformation of plant cells, particularly by means of acellular host according to the invention, this itself having beentransformed by a vector according to the invention, so as to integrate arecombinant sequence according to the invention within the genome ofthese cells,

[0045] as necessary, the production of transformed plants from theaforementioned transformed cells,

[0046] recuperation of recombinant lactoferrin, particularly humanlactoferrin, or derived proteins produced in said aforementionedtransformed cells or plants, particularly by extraction followed, asnecessary, by purification,

[0047] a genetically transformed plant, plant extract or part of aplant, particularly leaves and/or fruits and/or seeds and/or plantcells, characterised in that it contains one (or several) recombinantnucleotide sequence(s) according to the invention which is (are)integrated in a stable manner in the genome thereof, these plants beingparticularly chosen from amongst rape, tobacco, maize, peas, tomatoes,carrots, wheat, barley, potatoes, soy, sunflower, lettuce, rice, alfalfaand beets.

[0048] a lactoferrin, particularly a human lactoferrin, or a derivedprotein, characterised in that it is obtained by means of the method ofthe invention,

[0049] a genetically transformed plant, plant extract or part of aplant, particularly leaves and/or fruits and/or seeds and/or plantcells, characterised in that it contains a lactoferrin, particularly ahuman lactoferrin or a derived protein, according to the invention,these plants being particularly chosen from amongst rape, tobacco,maize, peas, tomatoes, carrots, wheat, barley, potatoes, soy, sunflower,lettuce, rice, alfalfa and beets.

[0050] the used of plants, plant extracts or parts of plants accordingto the invention, and/or proteins (lactoferrin, particularly humanlactoferrin, or derived proteins) according to the invention, for theproduction of pharmaceutical, medical, odontological, cosmetic orbiotechnological compositions,

[0051] a biomaterial or a pharmaceutical, medical, odontological,cosmetic or biotechnological composition characterised in that thiscomprises plants, plant extracts or parts of plants according to theinvention, and/or proteins (lactoferrin, particularly human lactoferrin,or derived proteins) according to the invention.

[0052] A pharmaceutical composition according to; the invention includesparticularly any composition according to the invention whichconstitutes or is used in the manufacture of a composition allowing forthe detection or treatment of a pathology or a symptom of bacterial,fungal or viral origin, an inflammation or a pathology having aninflammatory component, septic shocks, a pathology related to a cellulargrowth phenomenon or an iron deficit, such as anaemia.

[0053] A cosmetic composition according to the invention includesparticularly any composition according to the invention constituting (orused in the manufacture of) an additive for preparations (creams,ointments, makeup, salves).

[0054] Advantageously, the recombinant sequences according to theinvention contain one (or several) sequence(s) which code for a peptideresponsible for addressing recombinant polypeptides in a specificcompartment of the plant cell, particularly in the endoplasmic reticulumor in the vacuoles, or even outside the cell in the pectocellulosic wallor in the extracellular space know as the apoplasm.

[0055] Amongst the transcription terminators which can be used for thetransformation of plant cells within the framework of the presentinvention, examples include terminator polyA 35S of the cauliflowermosaic virus (CaMV), or the terminator polyA NOS, which corresponds tothe non-coding 3′ region of the nopaline synthase gene of the plasmid TIof the nopaline strain of Agrobacterium tumefaciens.

[0056] Accordingly, the invention takes as its object any recombinantnucleotide sequence such as described above which contains theterminator polyA 35S of the CaMV, or the terminator polyA NOS ofAgrobacterium tumefaciens downstream of said cDNA or a derived sequencethereof.

[0057] Amongst the transcription promoters which can be used for thetransformation of plant cells in the framework of the invention,examples include:

[0058] the promoter 35S (P35S), or advantageously the doubleconstitutive promoter (Pd35S) of the CaMV: these promoters allow forexpression of the recombinant polypeptides of the invention in theentire plant produced from transformed cells according to the invention,and are described in the article of Kay et al., 1987,

[0059] the promoter PCRU of the radish cruciferin gene allows for theexpression of the recombinant polypeptides of the invention in only theseeds (or grains) of the plant produced from transformed cells accordingto the invention and is described in the article by Depigny-This et al.,1992;

[0060] the promoters PGA1 and PGA6, which correspond to the non-coding5′ region of the seed reserve protein genes, GEA1 and GEA6 respectively,of Arabidopsis thaliana (Geubier et al, 1993) allow for specificexpression in seeds,

[0061] the chimeric promoter super-promoter PSP (Ni M. et al., 1995),which is constituted from a triple repetition of a transcriptionactivation element of the Agrobacterium tumefaciens octopine synthasegene promoter, of a transcription activation element of the mannopinesynthase gene promoter, and of the mannopine synthase promoter ofAgrobacterium tumefaciens,

[0062] the rice actin promoter followed the actin intron (PAR-IRA)contained in the plasmid pAct1-F4 described by McElroy et al., (1991),

[0063] the barley HMWG (high molecular weight glutenin) promoter(Anderson O. D. et al., 1989),

[0064] the maize γzein gene promoter (Pγzein) contained in plasmid pγ63described in Reina et al, (1990) which allows for expression in thealbumen of maize seed.

[0065] Accordingly, the object of this invention extends to anyrecombinant nucleotide sequence such as described above containing thedouble 35S constitutive promoter (Pd35S) of the CaMV, the promoter PCRUof the radish cruciferin gene, the promoters PGA1 or PGA6 of Arabidopsisthaliana, the chimeric promoter super-promoter PSP of Agrobacteriumtumefaciens, the rice PAR-IRA promoter, the barley HMWG promoter, or themaize Pγzein promoter, upstream of said cDNA or a sequence derivedtherefrom.

[0066] Sequences coding for an addressing peptide in the framework ofthe present invention may be of plant, human or animal origin.

[0067] Amongst sequences coding for an addressing peptide originating inplants, examples include:

[0068] the nucleotide sequence of 69 nucleotides (shown in the exampleswhich follow) which codes for the 23 amino acid prepeptide (signalpeptide) of sporamin A in sweat potatoes, this peptide signal allows therecombinant polypeptides of the invention to enter the secretion systemof the plant cells transformed according to the invention (that is tosay, principally in the endoplasmic reticulum),

[0069] the nucleotide sequence of 42 nucleotides (shown in the exampleswhich follow) which codes for the vacuolar addressing N-terminalpropeptide of 14 amino acids of sweet potato sporamin A, which allowsfor the accumulation of recombinant polypeptides according to theinvention in the vacuoles of plant cells transformed according to theinvention,

[0070] the nucleotide sequence of 111 nucleotides (shown in the exampleswhich follow) which codes for the prepropeptide of 37 amino acids ofsporamin A comprising the N-terminal part nearest the C-terminal partcomprising the 23 amino acids of the aforementioned signal peptide,followed by the 14 amino acids of the aforementioned propeptide: thisprepropeptide allows for entry of the recombinant polypeptides of theinvention in the secretion system, and their accumulation in thevacuoles of the plant cells transformed according to the invention; thethree aforementioned sequences are described in the articles of Murakamiet al., 1996, and Matsuoka et al., 1991,

[0071] the barley lectin carboxy terminal propeptide, particularly asdescribed in the articles of Schroder et al., 1993, and Bednarek et al.,1991,

[0072] and PRS (pathogenesis related protein, Corenlissen et al., 1986)which allows for secretion.

[0073] Amongst sequences which code for an addressing peptide, examplesalso include those coding for the peptides KDEL, SEKDEL and HDEL, andallowing for addressing in the endoplasmic reticulum.

[0074] The object of the invention also extends to any recombinantnucleotide sequence such as described above which contains a sequencecoding for all, or part, of a vacuole addressing peptide, particularlythat of sweat potato sporamin A, where this sequence codes for a vacuoleaddressing peptide which is located, in said recombinant nucleotidesequence, between the sequence coding for a signal peptide and thatcoding for said cDNA or a derived sequence thereof, in such a way thatthe first N-terminal amino acid of the vacuole addressing peptide isjoined to the last C-terminal amino acid of the signal peptide, andwherein the last C-terminal amino acid of said addressing peptide isjoined to the first N-terminal amino acid of the polypeptide coded bysaid cDNA or a derived sequence thereof, in the protein coded by saidrecombinant nucleotide sequence.

[0075] The object of the invention also extends to any nucleotidesequence such as described above which contains a sequence which codesfor all or part of a vacuole addressing peptide, particularly that ofbarley lectin, wherein this sequence codes for a vacuole addressingpeptide which is located upstream of the sequence coding for said cDNAor a derived sequence thereof, so that the first N-terminal amino acidof the vacuole addressing peptide is joined to the last C-terminal aminoacid of the polypeptide coded by said cDNA or a derived sequencethereof, in the protein coded by said recombinant nucleotide sequence.

EXAMPLE 1 Construction of the pBS-Lf Plasmid Containing the Entire cDNACoding for Human Lactoferrin

[0076] Lactoferrin is synthesised in the form of a pre-protein in whichthe sequence coding for the mature 692 amino acid protein is preceded bya 19 amino acid signal peptide (PSLf), having the sequenceMKLVFLVLLFLGALGLCLA.

[0077] The entire cDNA containing the signal peptide sequence was usedthis example. It was isolated from a human mammary gland cDNA bank(Clontech, item HL103L7b, CA, USA) constructed in the vector λgt11.Screening of the bank was carried out with an oligonucleotide probecorresponding to the amino acids of the signal peptide (that is to say,amino acids 1 to 19 of the immature hLf, also known as humanprelactoferrin) of Lf by means of the nitro-cellulose replicahybridsation method. Briefly stated, the clones are fixed in a NaOH 0.5M, NaCl 1.5 M bath for two minutes and then neutralised for 5 minutes ina NaCl 1.5 M, Tris-HCl 0.5 M, pH 7.4 solution. The filters are thenrinsed with 2×SSC (17.5 g/l NaCl, 8.8 g/l trisodium citrate, QSP 11).Lastly, the DNA is fixed by heat treatment at 80° C. for 2 hours. Thehybridisation was carried out using the oligonucleotide probe describedabove, labelled at ³²P, with 10⁶ cpm/ml Of hybridisation solution(6×SSC, 10× Denhardt, 0.1% (w/v) Nonidet NP40 (octylphenoxypolyethoxyethanol, Sigma), 100 μg/ml of salmon sperm DNA)overnight at 65° C. The ‘replicas were’ then washed 4 times in a 2×SSCsolution, 0.1% SDS at 42° C., then exposed.

[0078] By means of controlled hydrolysis of λgt11 by EcoRI (3 U/μg ofDNA for 2 min. at 37° C.) the entire Lf cDNA was cloned in the plasmidpBluescript SK (Stratagene, La Jolla, USA) at the EcoRI site, thusproducing the plasmid pBS-Lf.

EXAMPLE 2 Construction of a pBS-12 Plasmid Containing the Entire cDNACoding for Human Lactoferrin, Modified at its 3′ Extremity

[0079] The cDNA was modified at 3′ so as to be able to join the sequencesituated downstream of the stop codon with the terminator contained inpBIOC21, described below. Using PCR, this modification made it possibleto remove the natural EcoRI restriction site (GAATTC) located upstreamof the stop codon, and to create one, 9 base pairs downstream, followedby a XbaI site (TCTAGA).

[0080] This PCR on the pBS-Lf matrix was carried out using the followingset of oligodeoxynucleotides:

[0081] oligo 5′: 5′ ATGACAACACTGAGTGTCTGGCC 3′ (corresponding to nucleicacids 1991 to 2011, that is to say, to amino acids 644 to 671 on theimmature hLf)

[0082] oligo 3′: 5′ CCGTCTAGAGAATTCGTTTTACTTCCTGAGGGAGTTCAC 3′ whichcontains the mutation sites (oligonucleotide overlapping the stopcordon, corresponding to amino acid 711 of the immature hLf).

[0083] PCR Conditions:

[0084] The reagents used in the various PCRs were provided by Promega(Charbonnière, France). The oligodeoxynucleotides were synthesised byEurogentec (Seraing, Belgium). In each case, 5 ng of matrix wereincubated in the presence of 100 pmol of each of the twooligodeoxynucleotides, 3 μl of dNTP 10 nM, 6 μl of MgCl₂ 25 nM, 0.5, μl(2.5 U) of Taq DNA polymerase, for a final volume of 100 μl, includingthe supplier's buffer.

[0085] The PCR was carried out with a bio-med THERMOCYCLER 60 (B.Braun). The first denaturation took place for 5 minutes at 94° C. Thiswas followed by 30 cycles each comprising 1 minute at 94° C., 1 minuteat (Tm-10° C., that is 50° C.) and 1 minute at 72° C.

[0086] The product of the PCR contained a single NdeI site (amino acid678 of the immature hLf) of lactoferrin cDNA. This fragment washydrolysed by XbaI and NdeI and sub-cloned to pBS-Lf with the NdeI-XbaIfragment deleted, which resulted in the production of the plasmidpBS-12.

[0087] Ligation conditions were as follows: 100 ng of plasmid and 100 ngof inserts were incubated overnight at 16° C. in the presence of 4U ofT4 DNA ligase (Strategene) in the buffer and under the conditionsrecommended by the supplier, that is, in a final volume, of 50 μlcontaining, amongst other things, 1 μl of 10 mM ASP. These conditionswere used for all the ligation reactions described hereafter.

[0088] The product of the ligation was used to transform DH5α bacteria,which had been made competent beforehand using the Rubididum methoddescribed below.

[0089] Preparation of competent bacteria:

[0090] 1 ml of the night culture of DH5α was recultured in a LB medium(10 g/l bactotryptone (g/l of yeast extract, 5 g/l of NaCl) containingKCl (250 mM) and MgSO4 (16 mM) until a DO₆₅₀ of 0.3 was obtained. Thebacteria suspension was then centrifuged at 2500 rpm for 5 minutes at 4°C.; the pellet was resuspended in a Tfb1 solution (RbCl 100 mM, MnCl₂ 10mM glycerol 15%, pH 5.8 adjusted with HOAc 0.2 M) incubated on ice for15 minute, then centrifuged again under the same conditions. This time,the pellet was resuspeneded in 6.5 ml of buffer Ttb2 (MOPS 10 mM, RbCl10 mM, CaCl₂ 75 mM, glycerol 15%, pH 7 adjusted with NaOH 0.5 M). Thecells were then portioned at 200 μl per tube and stored at −80° C. forthe following transformation processes.

[0091] At the time of the transformation process, the coils were thawedon ice and the DNA solution (25 μl of the ligation reaction, that is 50ng of DNA) was added to (200 ml of competent DH5α. The suspension wasincubated on ice for 20 minutes then plunged into a bath a 42° C. for 90seconds before returning it to the ice for 5 minutes. The suspension wasrecultured in a liquid LB medium for 1 hour, then spread on a LB mediumcontaining the selection marker. Following one night of culturing at 37°C., the clones were analysed.

[0092] This method was used for all the successive transformations.

[0093] After analysing the pBS12 clones, the sequence of the fragment,which was amplified by means of PCR, was confirmed by sequencing (Sangeret al., 1975) of this plasmid (Sequenase DNA Sequencing Kit, UnitedStates Biochemical Corporation, Cleveland, USA).

EXAMPLE 3 Construction of the Plasmid pBS-14, Derived from pBS-12,Comprising the cDNA Coding for Human Lactoferrin wherein the SignalPeptide (PSLf) has been Replaced by that of the Signal Peptide of SweetPotato Sporamin (PSSp)

[0094] Firstly, a SalI site was introduced downstream of XhoI in thecDNA of Lf at the first codon of the mature protein sequence, so as tobe able to join this with the sequence coding the sweet potato sporaminsignal peptide (PSSp; Muralkami et al., 1986; Matsuoka et al., 1991)allowing, in theory, for secretion. The sweet potato sporamin signalpeptide comprises 23 amino acids in the sequence MKAFTLALFALSLYLLPNPAHS.

[0095] The oligodeoxynucleotides, oligo 5′:

[0096] 5′ TAACTCGAGGCCGGGTCGACGGAGAAGGAGTGTTCAGTG 3′ containing SalI(GTCGAC) and XhoI (CTCGAG); amino acids 16 to 28 of the immature hLf,and,

[0097] oligo 3′:5′ ACCCGTCCAATTCAAGAATGGACGAAG 3′ containing, XcmI(CCAATTCAAGAATGG, amino acid 153 of the immature hLf), were used forthis PCR on the pBS-12 matrix, the conditions for which are describedabove. The fragment produced by the PCR was hydrolysed by XhoI and XcmI.It was inserted at the XhoI and XcmI sites of pBS-12 using the ligationconditions described above. The transformation was carried out asdescribed above. The resulting plasmid was called pBS-13. The sequencethereof was verified by sequencing as described above.

[0098] Secondly, in order to join the cDNA of the signal peptide withthat of mature Lf, a SalI site (GTCGAC) was introduced by means of PCRat the last codon of the sporamin peptide sequence, as well as a XhoIsite (CTCGAG) followed by an EcoRI site immediately upstream of the ATGinitiation codon. These modifications were carried out using PCR on apMAT103 matrix, according to the conditions described above. Theoligodeoxynucleotides chosen allowed for the insertion of the followingrestriction sites:

[0099] oligo 5′: 5′ TCCCTCGAGGAATTCATGAAAGCCTTCACACTC 3′ and,

[0100] oligo 5′: 5′ TCCGTCGACCGGAATGGGCTGGATTGGGCAGC 3′

[0101] After digestion of the fragment, amplified by XhoI and Sal L,this was sub-cloned in pBS-13 which had been hydrolysed beforehand byXhoI and SalI. The resulting pBS-14 plasmid was verified by sequencingthe first 500 nucleotides of the chimeric protein.

EXAMPLE 4 Construction of pBIOC21

[0102] Expression of the cDNA coding for lactoferrin in tobacco leavesand seeds required the following regulatory sequences:

[0103] 1. the double 35 S constitutive promoter (Pd35S) of the CaMV(cauliflower mosaic virus). This consists of a duplication of thesequences which activate transcription and which are located upstream ofthe TATA element of the natural 35S promoter (Kay et al., 1987);

[0104] 2. the transcription terminator sequence, terminator polyA35(T35S), which corresponds to the non-coding 3′ region of thedouble-stranded circular DNA cauliflower mosaic virus producing the 35Stranscript (Franck et al., 1980).

[0105] The constructions of the various plasmids via recombinant DNAmethods derive from pBIOC4. The binary plasmid derives from pGA492 (An,1986). The plasmid deriving from pBIOC4 and containing the expressioncassette “PD35S-T35S” is the plasmid pBIOC21.

[0106] The various elements allowing for the reproduction of theseconstructions are, for example, included in the description of patentapplication WO9633277, which is incorporated by way of reference.

EXAMPLE 5 Construction of pBIOC21-PSLf-Lf

[0107] Plasmid pBS-12 was hydrolysed by EcoRI to isolate the 2160 basepair fragment corresponding to the sequence which codes for humanlactoferrin, preceded by its natural signal peptide. This fragment wassub-cloned in the binary plasmid pBIOC21. which had been EcoRIlinearised and dephosphorylated. The binary plasmid produced was calledpBIOC21-PSLf-Lf

[0108] The dephosphorylation reaction was applied to 800 ng of DNA inthe presence of 1U of CIP (Stratagene) in the supplier's buffer at avolume of 40 μl for 30 minutes at 37° C. The ligation and transformationreactions for the DH5α strain of Escherichia coli were carried out asmentioned above.

[0109] The plasmid DNA of pBIOC21-PSLf-Lf was introduced to the strainLBA4404 of Agrobacterium tumefaciens according to the Holsters (1978)method. The clones retained were verified by enzymatic digestion of theplasmid DNA introduced.

EXAMPLE 6 Construction of pBIOC21-PSSp-Lf

[0110] The DNA coding for the chimeric protein “PSSp-Lf” was excisedfrom pBS-14 by means of hydrolysis with EcoRI. This 2150 base-paircoding fragment was sub-cloned, at the dephosphorylated pBIOC21 EcoRIsite. The binary plasmid produced was called pBIOC21-PSSp-Lf.

[0111] The dephosphorylation, ligation and transformation reactions forthe DH5α strain of Escherichia coli were carried out as above.

[0112] The plamid DNA of pBIOC21-PSSp-Lf was introduced to the strainLBA4404 of Agrobacterium tumefaciens according to the Holsters (1978)method. The clones retained were verified by enzymatic digestion of theplasmid DNA introduced.

EXAMPLE Construction of pACT-IA-PSLf-Lf

[0113] Constitutive expression in maize seed required the followingregulatory sequences.

[0114] rice actin promoter followed by the rice actin intron (pAR-IAR)contained in the plasmid pAct1-F4, described by McElroy et al., (1991)

[0115] the transcription terminator sequence, terminator polyA NOS,which corresponds to the non-coding 3′, region of the nopaline synthasegene of the Ti plasmid of nopaline strain Agrobacterium tumefaciens(Depicker et al., 1982).

[0116] The plasmid pBSII-pAR-IAR-tNOS in which the coding sequence“PSLf-Lf” was introduced is described, for example, in patentapplication WO9633277, which is incorporated by reference.

[0117] The coding sequence “PSLf-Lf” was isolated from pBS-12 by meansof enzymatic digestion by Eco-RI. The digested fragment was purified bymeans of electrophoresis on 0.8% agarose gel, electro-eluted, alcoholprecipitated, dried, and resuspended in H₂O. It was treated with Klenowenzyme (New England Biolabs) according to the manufacturer'srecommendations. It was inserted in the plasmid pBSII-pAR-IAR-tNOS,double digested by SalI and NcoI, purified, treated with Mung BeanNuclease (New England Biolabs) and dephosphorylated by the veal alkalinephosphatase enzyme (Boehringer Mannheim) in accordance with therecommendations of the manufacturer. Ligation was carried out with 20 ngof the dephosphorylated vector and 200 ng of DNA fragments containingthe sequence coding four “PSLf-Lf”, described above, in a reactionsolution of 20 μl, in the presence of 2 μl of T4 DNA ligase×10 buffer(Amersham), 2 μl of 50% polyethylene glycol 8000 and 5 U of T4 DNAligase (Amersham) at 14° C. for 16 hours. DH5α strain Escherichia colibacteria which had been made competent beforehand were transformed(Hanahan, 1983). The plasmid DNA of the clones produced, which werescreened with 50 μg/ml ampicillin was extracted using the alkaline lysismethod (Birnboim and Doly, 1979) and analysed by means of enzymaticdigestion by restriction enzymes. The plasmid produced was calledpACT-IA-PSLf-Lf.

EXAMPLE 8 Construction of pACT-IA-PSSp-Lf

[0118] The construction of pACT-IA-PSSp-Lf is similar to thatpACT-IA-PSLf-Lf, except that the sequence “PSLf-Lf” is replaced withthat coding for “PSSp-Lf”, corresponding to the EcoRI fragment treatedwith the Klenow enzyme (New England Biolabs), isolated from pBS-14. Theresulting plasmid was named pACT-IA-PSSp-Lf.

EXAMPLE 9 Construction of pgzein-PSLf-Lf

[0119] Expression in the albumen of maize seed required the followingregulatory sequences:

[0120] the maize zein gamma (g) gene promoter (Pgzein) contained in theplasmid p63, described by Reina et al., 1990. Plasmid p63 is the resultof cloning Pgzein to replace the 35S promoter (P35S), at the HindIII andXbaI sites of plasmid pUC18, which contains, between its HindIII andEcoRI sites, the expression cassette “P35S-gus-TNOS” of pBI221, which ismarketed by Clontech. It allows for expression in the albumen of maizeseed.

[0121] the transcription termination sequence, terminator polyA NOS,which corresponds to the non-coding region of the nopaline strainAgrobacterium tumefaciens Ti plasmid nopaline synthase gene (Depicker etal., 1982).

[0122] The plasmid pgzein-PSLf-Lf where the sequence “PSLf-Lf” is placedunder the control of Pgzein was produced by cloning at the sites SadIand BamHI, treated by the enzyme T4 DNA polymerase (New EnglandBiolabs), then dephosphorylated by the veal alkaline phosphatase enzyme(Boehringer Mannheim) of plasmid p63, of the EcoRI fragment treated withKlenow (New England Biolabs) isolated from pBS-12. Ligation was carriedout as described in Example 7. The DH5α Escherichia coli bacteria, whichhad been made competent beforehand, were transformed (Hanahan, 1983).The plasmid DNA of the clones produced, which were screened with with 50μg/ml ampicillin, was extracted using the alkaline lysis method(Birnboim and Doly, 1979) and analysed by means of enzymatic digestionby restriction enzymes. The plasmid produced was called pgzein-PSLf-Lf.

EXAMPLE 10 Construction of pgzein-PSSp-Lf

[0123] The construction of pgzein-PSSp-Lf is similar to that ofpgzein-PSLf-Lf, except that the sequence “PSL-Lf” is replaced with thatcoding for “PSSp-Lf”, corresponding to the EcoRI fragment treated withthe Klenow enzyme (New England Biolabs), isolated from pBS-14. Theresulting plasmid was named pgzein-PSSp-Lf.

EXAMPLE 11 Construction of pHMWG-IA-PSLf-Lf

[0124] Expression in maize seed required the following regulatorysequences.

[0125] the wheat HMWG (high molecular weight glutenin) promoter(Anderson et al., 1989) followed by the rice actin intron,

[0126] the transcription terminator sequence, terminator polyA 35S(T35S), which corresponds to the non-coding 3′ region of thedouble-stranded circular DNA cauliflower mosaic virus producing the 35Stranscript (Franck et al., 1980). The pHMWG-IA plasmid results fromcloning PHMWG-IA in place of the promoter PD35S (P35S) of the plasmidpJIT163. The various elements allowing for this construction to bereproduced are, particularly, described in international patentapplication No. WO 96/33277.

[0127] The plasmid pHMWG-IA-PSLf-Lf where the sequence “PSLf-Lf” isplaced under the control of pHMWG-IA was produced by cloning at theEcoRI site, dephosphorylated by the veal alkaline phosphatase enzyme(Boehringer Mannheim), of plasmid pHMWG-IA, of the EcoRI fragmentisolated from pBS-12. Ligation was carried out with 100 ng of thedephosphorylated vector and 50 ng of DNA fragments containing thesequence coding for “PSLf-Lf”, described above, in a 10 μl reactionsolution, in the presence of 1 μl of T4DNA ligase tampon (Amersham) at14° C. The DH5α Escherichia coli bacteria, which had been made competentbeforehand, were transformed (Hanahan, 1983). The plasmid DNA of theclones produced, which were screened using 50 μg/ml ampicillin, wasextracted using the alkaline lysis method, and analysed by means ofenzymatic digestion by restriction enzymes. The plasmid produced wascalled pHMWG-IA-PSLf-Lf.

EXAMPLE 12 Construction of pHMWG-IA-PSSp-Lf

[0128] The construction of pHMWG-IA-PSSp-Lf is similar to that ofpHMWG-IA-PSLf-Lf, except that the sequence “PSLf-Lf” is replaced withthat coding for “PSSp-Lf”, corresponding to the EcoRI fragment isolatedfrom pBS-14. The resulting plasmid was named pHMWG-IA-PSSp-Lf.

EXAMPLE 13 Production of Transgenic Solanaceous Plants

[0129] A. Transformation of Tobacco Plants

[0130] The tobacco plants used for the transformation experiments(Nicotiana tabacum var. Xanthi NC) were cultivated in vitro on aMurashige and Skoog (1962) basic medium to which the vitamins of Gamborget al., (1968, Sigma item M0404), 20 g/l of saccharose, and 8 g/l ofagar (Merck) were added. The pH of the medium was adjusted to 5.8 with apotassium solution before autoclaving at 120° C. for 20 minutes. Thetobacco plantlets were replanted from intra-node cuttings each 30 daysinto this multiplication medium MS20 (M0404 at 4.4 g/l, saccharose at 20g/l, agar agar at 8 g/l, pH 5.7).

[0131] All of the in vitro cultures were carried out inclimate-controlled enclosures, under the following conditions:

[0132] luminous intensity of 30 μE m⁻².S⁻¹; photoperiod of 16 hours;

[0133] thermoperiod of 26° C. in the day and 24° C. at night.

[0134] The transformation method used was derived from that of Horsch etal., (1985).

[0135] A pre-culture of LBA4404 strain Agrobacterium tumefacienscontaining the binary plasmids was carried out for 48 hours at 28° C.,under agitation, in an LB medium to which suitable antibiotics(rifampicin and tetracycline) were added. The pre-culture was thendiluted by a factor of 50 in the same medium and cultivated under thesame conditions.

[0136] After one night, the culture was centrifuged (10 min., 3000 g),the bacteria were resuspended in an equivalent volume of liquid MS30medium (M0404 at 4.4 g/l, saccharose at 30 g/l, pH 5.7) and thissuspension was diluted by a factor of 10.

[0137] Explants of approximately 1 cm² were cut from the plantletsdescribed above. These were then placed in contact with the bacterialsuspension for 1 hour, then quickly dried on filter paper and placed ina coculture medium (solid MS30 consisting of liquid MS30 to which agaragar at 8 g/l, BAP 1 mgA and ANA at 0.1 mg/l had been added).

[0138] Two days later the explants were transferred to Petri dishes onan MS30 regeneration medium, (consisting of the coculture medium towhich kanamycin at 200 mgA and Augmentin at 400 mgA had been added).This medium contains a selection agent, kanamycin (200 mg/l), abacteriostatic, Augmentin (400 mg/l) and the hormones necessary toinduce buds (BAP, 1 mg/l and ANA, 0.1 mg/l). The explants were replantedin the same medium after 2 weeks of culture. After a further two weeks,the buds were replanted into Petri dishes in the development mediumconsisting of MS20, medium to which kanamycin and Augmentin had beenadded. Fifteen days later, half the buds were replanted. It takesapproximately 20 days for roots to form. At the end of this period, theplantlets can be cloned by way of inter-node cuttings, or can be put ina greenhouse.

[0139] B. Production of Transgenic Tomato Plants

[0140] cv. UC82B tomato seeds were sterilised with 10% Domestos for 15minutes and rinsed 3 times in sterile water. The last rinse is carriedout for 10 minutes, under agitation.

[0141] Seeds rinsed in this manner were allowed to germinate in a MSSV/2medium (basic Murashige and Skoog medium (1962, Sigma item M6899)/2 towhich the vitamins of Nitsch (Thomas and Praqtt, 1981), 30 g/l ofsaccharose, and 8 g/l of agar (Merck) were added, pH 5.9), for 7 to 8days in a climate controlled chamber (luminous intensity of 30 μEm⁻².S⁻¹; photoperiod of 16 h/8 h, 26° C.).

[0142] The transformation method used is derived from that of Fillattiet al., (1987).

[0143] A pre-culture of LBA4404 strain Agrobacterium tumefacienscontaining the binary plasmids was carried out for 48 hours at 28° C.,under agitation, in an LB medium to which suitable antibiotics(rifampicin and tetracycline) were added. The pre-culture was thendiluted by a factor of 50 in the same medium and cultivated under thesame conditions for one night. The DO at 600 nm was measured, theagrobacteria were centrifuged (10 min., 3000 g), resuspended in a liquidKCMS medium (described in the publication of Fillatti et al., 1987) soas to obtain a DO of 0.8 at 600 nm.

[0144] Technical improvements were made to certain processes within themethod of Fillatti et al., (1987). The pre-culture of the explants andthe coculture are as described by Filliatti et al, (1987) except thatacetosyringon (200 nM) is added to the KCMS medium.

[0145] The 2z washing medium differs in that cefotaxime is added at 500mg/l, in place of carbenicillin. The development medium used is composedof the basic Murashige and Skoog medium (1962, Sigma item M6899) towhich the vitamins of Nitsch, 20 g/l of saccharose, 50 mg/l ofkanamycin, 200 mg/l of Augmentin, 1 mg/l of ANA, and 0.5 mg/l of zeatinwas added.

EXAMPLE 14 Production of Transgenic Rape Plants

[0146] Spring rape seed (Brassica napus cv WESTAR or Limagrain strains)were disinfected for 40 minutes in a 15% Domestos solution. After 4rinses in sterile water, the grains were allowed to germinate in pots of7 cm in diameter and 10 cm in height with 7 seeds to a pot, in theMurashige and Skoog mineral medium (Sigma M 5519) with 30 g/l ofsaccharose, solidified with 5 g/l of agar gel. These pots were placed ina culture chamber at 26° C. with a photoperiod of 16 h/8 h and aluminous intensity in the order of 80 mm.

[0147] After 5 days of germination, the cotyledons were removed in asterile manner, cutting each petiole approximately 1 mm above thecotyledon node.

[0148] At the same time, a pre-culture of strain LBA4404 ofAgrobacterium tumefaciens containing the binary plasmid was prepared ina 50 ml Erlenmeyer flask, for 36 hours, in 10 ml of 2YT bacterialculture, to which the antibiotics used in the selection of the chosenstrain were added.

[0149] This pre-culture was used to seed a new bacterial culture at0.1%, prepared under the same conditions. After 14 hours, the culturewas centrifuged for 15 minutes at 3000 g, and the bacteria wereresuspended in an equivalent volume of liquid germination medium. Thissuspension was portioned into Petri dishes of 5 cm in diameter at 5ml/dish.

[0150] The cut-end of a petiole was immersed in the solution ofagrobacteria thus prepared for a few seconds, then the petiole waspushed a, few millimetres into the regeneration medium. This medium hasthe same basic composition as the germination medium but 4 mg/l ofbenyl-amino-purine (BAP), a phyto-hormone which encourages the formationof new buds was added. Ten explants (cotyledon with petiole) werecultured in each 9 cm diameter Petri dish (Greiner item 664102).

[0151] After two days of coculture under the same environmentalconditions as used for germination, the explants were replanted inPhytatray trays (Sigma, item P1552) containing the same medium asbefore, to which a selection agent: 45 mg/l of kanamycin sulphate(Sigma, item K4000) and a bacteriostatic: a mixture (by weight) of ⅙potassium clavulanate and ⅚ sodium amoxicillin (injectableAugmentin)were added at 600 mg/l.

[0152] Twice thereafter, at intervals of 3 weeks, the explants werereplanted in a sterile manner into new culture under the sameconditions.

[0153] The green buds which appear after two of three replantings wereseparated from the explant and cultured individually in transparent potsof 5 cm in diameter and 10 cm in height containing the same medium asabove, but lacking in BAP. After three weeks of culture, the stems ofthe transformed buds were cut and the buds were replanted in a pot offresh medium. After three or four weeks, the roots are well enoughdeveloped to allow for acclimatisation of the plantlet in a phytoron.The buds which were not green or do not have roots were eliminated Thesealants were then transplanted into square pots of 7×7 cm; filled withpotting soil (NF standard U4455 1: 40% brown peat, 30% sieved sol and30% sand) saturated with water. After two weeks of acclimatisation inthe pytotron (temperature 21° C., photoperiod 16 h/8 h and 84% relativehumidity), the plantlets were repotted in pots of 12 cm in diameter,filled with the same potted soil, enriched by timed-release fertiliser(Osmocote, 4 g/l of potting soil) then moved to a greenhouse (class S2)maintained at 18° C., with watering twice a day for 2 minutes perwatering.

[0154] As soon as flowers appear, these were bagged (Crispac, item SM570y 300 mm×700 mm) so as to prevent cross fertilisation.

[0155] When the siliquas reached maturity, these were gathered, dried,then threshed. The seeds obtained in this manner were used to measurebiochemical activity. Selection of transgenic descendants is carried outby germination in a medium containing 100 to 150 mg/l of kanamycinsulphate (depending on genotype). The operating conditions were the sameas those described above, with the exception of the fact that thegermination took place in glass tubes, with only one seed per tube. Onlythose plants which developed secondary roots within the first threeweeks were acclimatised in the phytotron before being moved to thegreenhouse.

EXAMPLE 15 Production of Transgenic Maize Plants

[0156] A. Production and Use of Maize Calluses as Objects of GeneticTransformation

[0157] Genetic transformation of maize, regardless of the method used(electroporation, Agrobacterium, microfibres, particle cannon) requires,in general, the use of cells which are not differentiated in rapiddivisions, and thus have conserved the ability to regenerate entireplants. This type of cell makes up the embryogenic friable callus (knowas type II) of maize.

[0158] These calluses were obtained from immature embryos of genotype H1II or (A188 x B73) according to the method and using the media describedby Armstrong (1994). The calluses obtained in this manner weremultiplied and maintained by successive replanting each 15 days, in theinitiation medium.

[0159] Plantlets were regenerated from these calluses, by modifying thehormonal and osmotic balance of the cells according to the methoddescribed by Vain et al., (1989). These plants are then acclimatised ina greenhouse, or they can be crossed or self fertilised.

[0160] B. Use of a Particle Cannon for the Genetic Transformation ofMaize

[0161] The pervious section describes the production and regeneration ofthe cell line required for transformation; a method of genetictransformation allowing for the stable integration of modified geneswithin the plant genome will be described here. This method is based onthe use of a particle cannon, and is the same as that described by J.Finer (1993). The target cells are callus fragments as described inparagraph 1. Four hours before bombardment, 16 fragments per dish ofthese fragments, having a surface area of 10 to 20 nm², were placed inthe centre of Petri dishes containing the same medium as theinitialisation medium, to which 0.2 M of mannitol+0.2 M of sorbitol wereadded. The plasmids bearing the genes to be introduced were purifiedwith a Qiagen column according to the manufacturer's instructions. Theywere then precipitated on tungsten particles (M10) according to themethod described by Klein (1987). Particles coated in this manner werefired at the target cells, using a cannon according to the methoddescribed by J. Finer (1992).

[0162] The dishes of calluses which had been bombarded in this mannerwere then sealed with Scellofrais, and cultivated in the dark at 27° C.The first replanting was performed 24 hours later, then every 15 daysfor a period of three months, using the same medium as the initiationmedium, to which a selection agent was added, the nature andconcentration of which varies with the gene used (see paragraph 3). Theselection agents which can be used generally consist of the activeingredients of certain herbicides (Basta™, Round Up™) or certainantibiotics (Hygromycin, Kanamycin, etc.).

[0163] After 3 moths, or sometimes earlier, the calluses which are notinhibited in terms of growth by the selection agent, normally, and inthe majority of cases, composed of cells resulting from the division ofa cell which has integrated one or more copies of the selection gene inits genetic heritage, appear., The frequency with which such callusesare obtained was approximately 0.8 calluses per bombarded dish.

[0164] These cells were identified, individualised, amplified and thencultivated, so as to regenerate plantlets (cf. Paragraph A, Example 15).In order to avoid all possible interference by non-transformed cells,all of these operations were carried out in culture media containing theselection agent.

[0165] The plants regenerated in this manner were acclimatised, thencultivated in greenhouses where they could be crossed orself-fertilised.

[0166] The plants obtained according to the invention have a phenotypedistinct from that obtained in. WO9637094.

EXAMPLE 16-a Detection of Recombinant Human Lactoferrins (rhLf) fromPlant Leaves

[0167] In order to test for the most productive plants, soluble proteinswas carried out on twenty transformation products for each of theconstructions, pBIOC21-PSLf-Lf and pBIOC21-PSSp-Lf, followed bydemonstration of the quality and quantity of the recombinant Lf (rhLf)present. The leaves were ground in, liquid nitrogen until a fine powderwas obtained. The soluble proteins were extracted from the vegetalmatter using an extraction solution (Tris-HCl1 50 nM, EDTA 1 mM, NaCl100 mM, Triton X 100 0.1%, pH 6.5) at a ratio of 4 ml of solution pergram of leaf. After centrifuging the extract for 30 minutes at 13 000 g,the supernatant was decanted, filtered and used for the followingprocesses in the demonstration of the presence of recombinantlactoferrin.

[0168] ELISA Method of Lactoferrin Detection (Micogami et al., 1994)

[0169] Selection of a transformant for each type of molecularconstruction was carried out by comparing the quantity of recombinant Lfwith the quantity of soluble proteins. Quantification of solubleproteins was performed using microtitration (Promega, Charbonnière,France), and the rate of expression of rhLf was measured using the ELISAmethod described hereafter.

[0170] A polyclonal rabbit anti-human lactoferrin antibody was producedas follows: immunisation of the rabbit was produced by three successiveintramuscular injections of hLf For the first injection, 1 mg of hLfdissolved in 0.25 ml of nonpyrogenic physiological serum and 0.25 ml ofcomplete Freund adjuvant (Difco) were injected. Two weeks later, asecond injection was performed with 1 mg of hLf dissolved in 0.25 ml ofnonpyrogenic physiological serum and 0.25 ml of incomplete Freundadjuvant (Difco). Four weeks later, a third injection was performedunder the same conditions as the second. The first blood sample wastaken 10 to 15 days after the third injection and produced 20 ml ofblood. Thereafter, injections corresponding to booster shots (sameconditions as the second injection, described above) allowed for samplesto be taken every 16 days. The immunoglobulins of the rabbit antiserumwere purified by means of 35% saturated ammonium sulphate precipitation.The precipitate was dialysed in a 10 mM pH 8 TrisHCl buffer thenpurified by DEAE Trisacryl (IBF) chromatography.

[0171] The polyclonal antibodies were incubated in the wells of themicrotitration plate, at 100 μl per well (that is, 35 μg/ml), in asodium bicarbonate buffer (NaHCO3) 10 mM (pH 9.6) overnight at 4° C. orfor 2 hours at 37° C.

[0172] Incubation of the ELISA plate (Falcon) in 150 μl of PBS (NaCl 150mM, sodium phosphate (Na2HPO4, NaH2PO4) 50 mM, pH 7.5)—Tween 2% for 20minutes at room temperature allowed for blocking of the non-specificsites. Following three washes with PBS—Tween 0.05%, 100 μl of eachsample resulting from the soluble protein extracts was incubated at 37°C. for 2 hours, following which hLf was detected by the anti-Lfmonoclonal antibodies. The anti-Lf monoclonal antibodies were producedby fusing mouse splenocytes and SP₂O/Ag myeloma cells. The supernatantfrom the hybrid culture medium was used directly in the ELISA test. TheAb-rhLf complex was recognised by caprine anti-mouse antibodies labelledwith peroxidase (Pasteur Diagnostics) diluted to {fraction (1/3000)} inPBS and was placed in contact, as above, for 2 hours at 37° C. Detectionwas carried out under agitation by addition of the substrate solution (4mg of ortho-phenylenediamine dihydrochloride in 10 μl of 0.2 mM, pH 5.5“citric acid/sodium citrate” buffer in the presence of 10 μl of H₂O₂),coloration was achieved in a few minutes and the reaction was stopped byadding 50 μl of 20% H₂SO₄ per well. Optical density was read at 490 nM.

[0173] Between each step, the wells were washed with 0.05% PBS-Tween.

[0174] The ratio of rhLf to soluble proteins is shown in FIG. 1. Thisresulted in the selection of transformant T19 for the constructionpBIOC21-PSLfLF, and transformant T30 for the constructionpBIOC21-PSSp-Lf.

[0175] Analysis of the transformants by the ELISA method also showed arate of expression of rhLf in the T30 transformant obtained with theconstruction pBIOC-PSSp-Lf which was 3 times greater than that of thetransformant T19, transformed with pBIOC-PSLf-Lf This result indicates ahigher level of expression of fhLf when the protein's natural signalpeptide is replaced with that of sporamin.

[0176] Western Blot Method

[0177] In order to verify the apparent molecular mass of the rhLf of theselected transformants, Western Blot (FIGS. 2 and 3) andimmunoprecipitation (FIG. 4) tests were carried out using the solubleprotein extracts.

[0178] Following electrophoresis using 7.5% SDS-PAGE polyacrylamide gel(20 μg of soluble proteins per well), the proteins were transferred to anitro-cellulose membrane. This was then incubated in 2% *PBS-Tween, thenwashed 3 times with 0.05% PBS-Tween. Rabbit polyclonal antibodies wereproduced by means of purification of a rabbit antiserum by DEAE,Trisacryl chromatography. Following dilution to 5000 in PBS (that is 7μg/ml), these were placed in contact with the membrane for 3 hours at20° C. Thereafter this was washed with 0.05% PBS-Tween. The conjugate, acaprine anti-rabbit IgG (Pasteur Diagnostics) labelled with peroxidaseand diluted to 2500° in PBS, was then incubated for 1 hour at 20° C. Themembrane was washed three times with PBS again. Final detection wasperformed by incubating the membrane one last time in 100 ml of PBS inthe presence of 40 mg of DAB (3-3′-diaminobenzidine tetrahydrochloride)and 200 μl of H₂O₂.

[0179] *PBS: 150 mM NaCl, 50 mM Na2HPO4, NaH2PO4, pH 7.5.

[0180] As shown in FIG. 2, analysis of plants produced by the firsttransformation shows a band of an apparent molecular mass of 80 kDarecognised by the anti-Lf antibody. No signal was detected for thenon-transformed control, that is Nicotiana tabacum var, Xanthi. Thepresence of rhLf1 was demonstrated in the transformant t19, whichconfirms the results obtained by ELISA analysis.

[0181]FIG. 3 shows a Western Blot of the soluble proteins produced fromtransformant T30. It demonstrates the presence of rhLf of an apparentmolecular mass of 80 kDa which comigrates with Lf isolated from humanmilk. It should be noted that the rhLf appears as a double band. Thisdoublet is not explained by a modification at the N-terminal sequencelevel (cf. Example 2).

[0182] Immunoprecipitation

[0183] 6 mg of Sepharose A-Protein (Parmacia) were incubated for 1 hourat room temperature in the presence of rabbit polyclonal anti-human Lfantibodies. These antibodies were produced as described in Example 16.The Sepharose A-Protein beads were recovered by centrifuging, washed 3times with TBS (Tris-Hcl 20 mM, NaCl 150 mM, pH 8.2) and agitated in 20ml of soluble protein extract for 2 hours at room temperature. Following3 washings in TBS, the protein complex was disassociated in a recoverysolution (Tris 62.5 mM, SDS 2%, saccharose 10%, β-mercaptoethanol 5%,bromophenol blue 5%), separated with 7.5% SDS-PAGE polyacrylamide gel,and then coloured with Coomassie blue.

[0184]FIG. 4 shows that, following immunoprecipitation of the solubleprotein extracts of transformant T19, rhLf appears in the form of adoublet of an apparent molecular mass of 80 kDa. The same result isfound for transformant T30.

EXAMPLE 16-b Detection of Recombinant Human Lactoferrin (rhLf) in MaizeSeed

[0185] A. Extraction of Proteins from Maize Seed

[0186] Maize seed was ground in liquid nitrogen. Thereafter, this wassoaked in 5 ml of buffer (Tris-Hcl 100 mM pH 8, EDTA 1 mM, DTT 1 mM,NaCl 250 mM, Triton ×100 0.2%) per 500 mg of ground product overnight at4° C. The homogenate was then centrifuged at 10000 g at 4° C. for 10minutes. Quantification of the proteins was carried out according to theBradford method.

[0187] B. Immunodetection of Lactoferrin

[0188] The extracted proteins were denatured by heating to 95° C. for 5minutes in the presence of Tris-HCl 50 M pH 6.8, SDS 4%, saccharose 20%,β-mercaptoethanol 1%, and bromophenol blue 0.01%. The proteins were thenseparated by electrophoresis on polyacrylamide gel under 10% denaturedconditions. After migration, the proteins were transferred to anitro-cellulose membrane. Lactoferrin was detected using a humanlactoferrin antibody produced by rabbits, then a rabbit anti-IgG coupledto alkaline phosphatase.

[0189] C. Screening of Maize Seed Transformed with the ConstructionpHMWG-IA-PSSp-Lf

[0190] Amongst the 10 transformants tested, 5 present a positive signalfor immunodetection.

EXAMPLE 17 Example of Purification of Recombinant Human Lactoferrin(rhLf)

[0191] The rhLf1 from pBIOC21-PSLf-Lf and the rhLf2 from pBIOC21-PSSp-Lfwere purified by means of affinity chromatography using BrCN activated4B Sepharose gel (Pharmacia Biotech) to which an anti-lactoferrinpolyconal antibody had been joined.

[0192] 1 ml of gel, fixing approximately 3 mg of antibody, was batchincubated overnight at 4° C. in 100 ml of protein extract, the pH ofwhich had been adjusted to 8.2 using 1 M Tris.

[0193] Thereafter, the gel was thoroughly washed with TBS pH 8.2(Tris-HCl 20 mM, NaCl 150 nM, pH 8.2) followed by 1 M NaCl, pH 8.2 TBS.Elution was carried out with a glycine-HCl 0.2 M, pH 2.4 buffer, inthree successive elutions which were analysed on 7.5% polyacrylamidegel.

[0194] This method was used for the preparation of rhLf1, with a view toanalysis of the N-terminal sequence. Analysis with 7.5% SDS-PAGEpolyacrylamide gel of the rhLf1 and rhLf2 purified by this method showsno difference in mass between the two rhLf (FIG. 5).

[0195] For the chimeric protein resulting from pBIOC21-PSSp-Lf, a largerscale purification was carried out with a view to deeper analysis ofthis protein. Using ion exchange chromatography with an SP SepharoseFast Flow (Pharmacia Biotech) column, equilibrated with an acetatesolution of Na 0.2 M pH 7, and a Biopilot type apparatus (Pharmaciabitotech), rhLf2 was eluted with an NaCl gradient of 0 to 1 M. The rhLf2was found primarily in a fraction eluted at 0.7 M NaCl, as proved by theELISA tests carried out on all of the fractions collected.

[0196] The positive fractions were concentrated by means ofultrafiltration with Centriprep 30 (Amicon, Bevedrly, USA), dialysedagainst PBS (NaCl 50 m, Na2HPO4-NaH2PO4 50 mM, pH 7.5) and fozen at −20°C. The purity of the rhLf2 was verified using 7.5% SDS-PAGEpolyacrylamide gel.

EXAMPLE 18 Variant form of Extraction and Purification of RecombinantHuman Lactoferrin

[0197] Fresh or frozen tobacco leaves were ground in liquid nitrogenuntil a fine powder was obtained. The soluble proteins were thenextracted from the vegetal matter using an extraction solution (Tris/HCl50 mM, NaCl 100 mM, EDTA 1 mM, dithiothreithiol 1 mm, Triton X10 0.2%(w/v), phenylmethylsulphonyl fluoride 1 mm, pH 7.0) containing 0.2 g ofDiaion Sepabeads SP 825 (Resindion) or Diaipn HP 20 (Supelco) per ml ata rate of 4 ml of solution per gram of vegetal material. The groundproduct was incubated at 4° C. for 12 hours with magnetic stirring. Thepurpose of this step is to extract the soluble proteins and to absorb aportion of the vegetal pigments with the hydrophobic resin. The extractwas centrifuged for 45 minutes at 15000 g, then the supernatant wasfiltered with a 0.45 micron Millipore membrane.

[0198] The vegetal extract was subjected to chromatography using amono-S HR10-10 column (Pharmaci Biotech) equilibrated by a 0.2 M pH 7.8solution of sodium acetate, using a Biopilot type apparatus (PharmaciaBiotech). After passing the extract through the column, a NaCl gradientof 0 to 1 M in the 0.2 M sodium acetate was established in the columnfor 60 minutes, and the rhLf was found in an eluate fraction at 0.8NaCl. This fraction was frozen at −20° C.

EXAMPLE 19 Analysis of the N-Terminal Sequence

[0199] Analysis of the N-terminal sequence using the Edman degradationprocess showed that, for both of the constructions, that is to say, forthe proteins rhLf1 and rhL2, the sequence of the first 7 residues is“GRRRRSV”, which corresponds exactly to the N-terminal end of thereference mature human lactoferrin. These results show that joining thesignal peptide for sweet potato sporamin secretion or for that of humanlactoferrin to the sequence coding for mature human lactoferrin allowsfor correct cleavage of this signal peptide.,

EXAMPLE 20 MALDI/TOF Mass Spectrometry Analysis

[0200] Analysis was performed using a MALDI Vision 2000 laser desorptionmass spectrometer (Funnigan MAT, Brenen, Germany). 3 μl of a solutioncontaining 100 pmol of lactoferrin were added to 17 μl of matrixsolution containing 10 mg/ml of 2.5 dihydroxybenzoic acid in awater/acetonitrile (30/70) mixture. 1 μl of this product was sent to thetarget at the same time as a mass calibre (human serotransferrin, Sigma,with a molecular mass of 79590 Da in sinapinic acid). The molecular massof rhLf2 was 81250 Da, which corresponds to the mass calculated for thepolypeptide chain of hLf (76320 Da) to which two glycans of a total massof 5180 Da are attached. Based on these values, the glycan ratiorepresents 6.35% of the total mass of the glycoprotein.

[0201] Analysis of the glycoprotein monosaccharides was performed usinggaseous phase chromatography with a OV 101 capillary column, and byGirdel 300 chromatography. The helium flow was 10 ml/min and thepressure was 0.5 bars. The temperature was set to 120° C. to 240° C. at2° C./min. The trimethylsilylated derivatives were prepared bymethanolysis (Zanetta et al., 1992), by N reacetylation andtrimethylsilyation (Kamerling et al., 1975). Analysis shows a ratio of6.5% of total oses in the glycoprotein. The molar composition isdescribed below. N-acetyl glucosami neuramini Fucose Galactose Mannosene Xylose c acid rhLf2 1.5 0.7 3.0 3.3 0.7 0.0 Lfh 1.3 2.1 3.0 4.0 0.01.8

[0202] The results obtained suggest rhLf2 glycans with a structure ofthe type N-acetyl-lactosamine, with a certain heterogeneity in themonosaccharide molar composition. Of particular note is the presence ofxylose, the low level of galactose and the absence of N-acetylneuraminic acid.

EXAMPLE 22 Jurkat Cell Binding Experiments

[0203] Many studies carried out on lymphocytes have shown the presenceof specific receptors for lactoferrin on their surfaces. Theirappearance at lymphocyte surfaces has been studied using the mitogenagent, phytohemagglutinin (PHA, Mazurier et al., 1989). This work showedthat quiescent lymphocytes do not have receptors for lactoferrin andthat phytohemagglutinin induces the emergence of high affinity receptorson the surfaces of circulating lymphocytes, these receptors beingsynthesised during the first two days of activation. The bindingconstants and number of binding sites are 80 nM and 200000 nMrespectively under PHA activation conditions. This same receptor wasrecently studied for the Jurkat lymphoblastic strain which allows forstable and reproducible results (Bi et al., 1994). In the rhLf2 bindingtests, this Jurkat cell strain was used for testing the biologicalactivity of the recombinant protein.

[0204] The T lymphoblastic cells of the Jurkat strain were cultivated ina RPMI 1640 medium (GIBCO, Cergy Pontoise, France) pH 7.4 containing 25mM of Hepes, 2 mM of L-glutamine, gentamicin (5 mg/ml) in the presenceof 10% foetal veal serum which had previously been deactivated by heatin an oven with 5% CO₂ at 37° C.

[0205] At the subconfluence stage the cells were diluted to a density of4.10⁵/ml for the binding experiments.

[0206] 100 μg of rhLf was labelled with 0.2 m Ci of ¹²⁵I using iodobeads(Pierce, Rockford, USA) according to the recommendations of themanufacturer. The free iodine was removed by means of gel filtration,using a Sephadex G-25 column equilibrated with PBS (50 mM NaCl, 50 mMNa2HPO4-NaH2PO4,k pH 7.5). The binding experiments were carried out inRPMI containing 0.4% (w/v) of human transferrin, so as to avoidnon-specific rhLf2 binding on the cells or on the plastic. Aliquots of100 ml containing 5.10⁵ cells were separated into 1.5 propylene tubes inthe presence of labelled rhLf2 at concentrations ranging from 0 to 100mM.

[0207] Non-specific binding was evaluated in the presence of an 100molar excess of unlabelled Lf. The cells were incubated with theproteins for 1 hour at 4° C. in the presence of 0.01% (w/v) of sodiumazide. Lastly, the cells were washed 3 times in 0.5 ml of PBS.Radioactivity was then measured with a Compugamma gamma radiationcounter, LKB—Wallac (Turku, Finland).

[0208] Binding of rhLf2 labelled with ¹²⁵I to Jurkat T lymphoblasticcells was analysed using the Scatchard method (1949). As shown in FIG.6, the binding curves for rhLf2 and Lfh isolated from milk are similar.With the range of Lfh used, only one class of receptor was detected onthe cell surface, having a dissociation constant of 80.27±33 nM and anumber of sites per cell of 124400±36000. On the same cells, hLf bindswith a dissociation constant of 89.7±22 nM and a number of sites percell of 103300±14000.

[0209] Thus, the binding test shows that rhLf2 has a configuration veryclose to that of human milk Lf, as it is recognised with the sameparameters on its lymphocytic receptor.

EXAMPLE 23 Binding of rhLf2 on the Surface of HT29 Cells

[0210] Binding of human milk lactoferrin was demonstrated with aenterocytic strain (HT-29) derived from a human colic adenocarcinomacapable of enterocyte differentiation (Mikogami et al., 1994, 1995).This author showed that binding of lactoferrin on its enterocyticreceptor is not linked to the degree to which it is saturated in iron,and is specific, that is to say, it does not result from electrostaticor lectin interactions.

[0211] The number of sites on HT29 type cells is in the order of 3.10⁶per cell and the Kd is approximately 10-⁶ M. The HT-29-18C1 cloneconforms to these characteristics and was used for the binding of rhLf2,so as to verify its biological activity.

[0212] The HT29 cells of the 18C1 clone were cultivated in DMEM(Eurobio, Les Ullis, France) containing 2 mM of L-glutamine andgentamicin (5 mg/l) in the presence of 10% foetal veal serum which hadpreviously been deactivated by heat in an oven with 10% CO₂ at 37° C. Atthis subconfluence stage, the cells were divided and returned to culturein 2 cm² wells, with 2.10⁴ cells/cm². Following 21 days of culture,which allowed for enterocyte differentiation, these cells were used forbinding tests.

[0213] The rhLf2 was labelled with ¹²⁵I as described in example 19. Thecells in each well were incubated for 1 hour on ice, in the presence of0.01% (w/v) of sodium azide and human transferrin, which preventsnon-specific binding by the lactoferrin. The radioactivity of a 50 μldose was measured for calculation of the concentration of free ligandspresented to the cells. Following 5 rinses with 500 μl of DPBS+(Sigma,ref D1283), the cells were released by 200 μl of DPBS+/EDTA (5 g/l) andthe radioactivity of the rhLf2 bound was measured by a Compugamma λradiation counter, LKB-Wallac.

[0214] Binding of ¹²⁵I labelled rhLf2 to HT-29-18C1 enterocytic cellswas analysed by the Scatchard method. As shown in FIG. 7, the bindingcurves for rhLf2 and Lf isolated from milk are similar. With the rangeof lactoferrins used, only one class of receptors was detected on thesurface of the cells, having a dissociation constant of 0.8±0.19 μM, and1.8×10⁶+0.28×10⁶ sites per cell. On the same cells, human lactoferrinbinds with a dissociation constant of 1±0.2 μM and 4×10⁶±0.5×10⁶ sitesper cell.

[0215] This binding to enterocytic cells shows, as in Example 19, thatrhLf2 is recognised by the specific lactoferrin receptor in the same wayas the native protein is, which suggests good correspondence and abiological activity comparable to that of lactoferrin isolated frommilk.

EXAMPLE 24 Production of Bovine Lactoferrin by Plants and Purificationthereof

[0216] Methods such as those described in the invention can also be usedfor the production of bovine lactoferrin in plants.

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DESCRIPTION OF THE DRAWINGS

[0265]FIG. 1: Rate of expression of rhLf1 as a function of the totalquantity of soluble proteins (given as %). Columns 1 to 19: primarytransformants. Column NT: non-transformed tobacco.

[0266]FIG. 2: Detection of rhLf1 by Western Blot method using an extractof the soluble proteins of the primary transformants. Lanes 1, 2, 18,19, 20: primary transformants. Lane NT: non-transformed tobacco. Thepositions of the molecular mass controls are shown on the left.

[0267]FIG. 3: Detection of rhLf2 by the Western Blot method using aconcentrated extract of soluble proteins from transformant T30. Lane (1)analysis of transformant T30. (2) Human Lf, isolated from milk. Thepositions of the molecular mass controls are shown on the left.

[0268]FIG. 4: Detection of rhLf1 by immunoprecipitation using an extractof the soluble proteins of transformant T19. The SDS-PAGE analysis showsthe rhLf1 and the anti-Lf antibodies (Ab). The positions of themolecular mass controls are shown on the left.

[0269]FIG. 5: Analysis of rhLf1 and rhLf2 after purification by means ofaffinity chromatography.

[0270] Lane (1): rhLf1; lane (2): rhLf2. The positions of the molecularmass controls are shown on the left.

[0271]FIG. 6: Specific binding of rhLf2 (▴) and milk hLf (◯) on Jurkatlymphoblastic cells. The lactoferrins are labelled with ¹²⁵I.Non-specific binding is measured in 100 molar excess of non-labellednative Lf and represents approximately 25% of total binding. Thesevalues were determined by two separate experiments, both of which werecarried out twice.

[0272]FIG. 7: Specific binding of rhLf2 (▴) and milk hLf(◯) on HT29enterocytic cells. The lactoferrins are labelled with ¹²⁵I. Non-specificbinding is measured in the presence of 100 molar excess of non-labellednative Lf, and represents approximately 25% of total binding. Thesevalues were determined by two separate experiments, each of which wascarried out twice.

1. The use of a recombinant nucleotide sequence containing both: a cDNAcoding for a lactoferrin; particularly for mammalian lactoferrin,preferably bovine, porcine, caprine or human lactoferrin, or derivedproteins; and the elements required by a plant cell to produce thelactoferrin or derived proteins coded by said cDNA, particularly atranscription promoter and terminator recognised by the transcriptionalmachinery of plant cells, so as to transform these plant cells, so as toproduce from these cells, or from plants produced therefrom; lactoferrinor derived proteins.
 2. Use of a recombinant nucleotide sequenceaccording to claim 1, characterised in that the nucleotide sequencecontains a sequence which codes for a signal peptide responsible for thesecretion of recombinant polypeptides.
 3. Use of a recombinantnucleotide sequence according to claim 2, characterised in that thesignal peptide used is the signal peptide of sweet potato sporamin A. 4.A recombinant nucleotide sequence characterised in that it contains boththe sequence coding for a lactoferrin, particularly for mammalianlactoferrin, preferably bovine, porcine, caprine or human lactoferrin,or derived proteins; and the elements required by a plant cell toproduce the lactoferrin or derived proteins coded by said sequence,particularly a transcription promoter and terminator recognised by thetranscriptional machinery of plant cells.
 5. A recombinant nucleotidesequence according to claim 4, characterised in that it contains asequence which codes for a signal peptide responsible for the secretionof recombinant polypeptides.
 6. A recombinant nucleotide sequenceaccording to claim 5, characterised in that the nucleotide sequence usedis the signal peptide of sweet potato sporamin A.
 7. A vector,particularly a plasmid, containing a nucleotide sequence according toone of claims 4 to 6 inserted, as necessary, at a site which is notessential for its replication.
 8. A cellular host, particularly anybacteria such as Agrobacterium tumefaciens transformed by a vectoraccording to claim
 7. 9. A method of producing lactoferrin, particularlyhuman lactoferrin, or derived proteins, characterised in that itcomprises: transformation of plant cells, particularly by means of acellular host according to claim 8, this itself having been transformedby a vector according to claim 7, so as to integrate a recombinantsequence according to claims 4 to 6 within the genome of these cells; asnecessary, the production of transformed plants from the aforementionedtransformed cells; recovery of the recombinant lactoferrin, particularlyhuman lactoferrin, or derived proteins produced in said aforementionedtransformed cells or plants, particularly by extraction followed, asnecessary, by purification.
 10. A genetically transformed plant, plantextract or part of a plant, particularly leaves and/or fruits and/orseeds and/or plant cells, characterised in that it contains one (orseveral) recombinant nucleotide sequence(s) according to claims 4 to 6which is (are) integrated in a stable manner in the genome thereof,these plants being particularly chosen from amongst rape, tobacco,maize, peas, tomatoes, carrots, wheat, barley, potatoes, soy, sunflower,lettuce, rice, alfalfa and beets.
 11. A lactoferrin, particularly ahuman lactoferrin, or a derived protein, characterised in that it isobtained by means of the method of claim
 9. 12. A geneticallytransformed plant, plant extract or part of a plant, particularly leavesand/or fruits and/or seeds and/or plant cells, characterised in that itcontains a lactoferrin, particularly a human lactoferrin or a derivedprotein according to the invention, these plants being particularlychosen from amongst rape, tobacco, maize, peas, tomatoes, carrots,wheat, barley, potatoes, soy, sunflower, lettuce, rice, alfalfa andbeets.
 13. Use of plants, plant extracts or parts of plants according toclaim 10 or 12, and/or proteins (lactoferrin, particularly humanlactoferrin, or derived protein) according to claim 11, for theproduction of pharmaceutical, medical, odontological, cosmetic orbiotechnological compositions.
 14. A biomaterial or a pharmaceutical,medical, odontological, cosmetic or biotechnological compositioncharacterised in that this comprises plants, plant extracts or parts ofplants according to claim 10 or 12, and/or proteins (lactoferrin,particularly human lactoferrin, or derived protein) according to claim11.